The invention relates to the chemistry of biologically active compounds, namely, to a new method to prepare water-soluble porphyrin derivatives, particularly chlorin, bacteriochlorin, pheophorbide and bacteriopheophorbide derivatives of types 1 and 2. The compounds of the present invention can be used as photosensitizers for the photodynamic therapy of cancer, infections and other diseases as well as for light irradiation treatments in other cases. 
Wherein B is a ring having the structure: 
Wherein:
R1xe2x95x90xe2x80x94CHxe2x95x90CH2, xe2x80x94CH(OAlk)CH3, xe2x80x94CHO, xe2x80x94C(O)CH3, xe2x80x94CH2CH3, xe2x80x94CH(Alk)CH(COAlk)2, xe2x80x94CH2CH(COAlk)2, xe2x80x94CH(Alk)CH2COAlk, xe2x80x94CH(Alk)CH2CH(OH)CH3, and xe2x80x94CH2CH2CH(OH)CH3 
R2xe2x95x90xe2x80x94CH3, xe2x80x94CHO, xe2x80x94CH(OH)Alk, xe2x80x94CHxe2x95x90CHAlk, CH2OH, and CH2OAlk;
R3xe2x95x90xe2x80x94OH, xe2x80x94OAlk, xe2x80x94NH-Alk, NHxe2x80x94Xxe2x80x94COOxe2x88x92(HG)+, xe2x80x94NHxe2x80x94Yxe2x80x94NR8R9, -and NHxe2x80x94Yxe2x80x94OH;
R4xe2x95x90xe2x80x94OAlk, xe2x80x94NH-Alk, and NHxe2x80x94Xxe2x80x94COOxe2x88x92(HG)+;
R5xe2x95x90xe2x80x94OAlk, xe2x80x94NH-Alk, and NHxe2x80x94Xxe2x80x94COOxe2x88x92(HG)+;
R6xe2x95x90H and xe2x80x94COOAlk;
R7xe2x95x90xe2x80x94Oxe2x88x92(HG)+, xe2x80x94OAlk, xe2x80x94NH-Alk, and xe2x80x94NHxe2x80x94Xxe2x80x94COOxe2x88x92(HG)+;
R8xe2x95x90H and Alk
R9xe2x95x90H and Alk
Wherein:
xe2x80x94NHxe2x80x94Xxe2x80x94COOxe2x88x92=the residue of organic amino acid;
X=alkylidene, peptides, oligopeptides and xe2x80x94(CH2CH2O)nCH2CH2xe2x80x94, wherein n=1-30;
Y=alkylidene and xe2x80x94(CH2CH2O)nCH2CH2xe2x80x94, wherein n=1-30;
G=a hydrophilic organic amine (f.ex. N-methyl-D-glucamine and other amino-group containing carbohydrate derivatives, TRIS, amino acids, oligopeptides); and
Alk=an alkyl substituent.
Photodynamic therapy (PDT) is one of the most promising new techniques now being explored for use in a variety of medical applications (Photodynamic therapy, basic principles and clinical applications. Eds. B. W. Henderson, Th. J. Dougherty, Marcel Dekker, 1992, New York), and particularly is a well-recognized treatment for the destruction of tumors (Photodynamic tumor therapy. 2nd and 3rd generation photosensitizers. Ed. J. G. Moser, Harwood Academic Publishers, 1998, Amsterdam). Porphyrins are compounds widely used in PDT. A major problem in the pharmaceutical application of porphyrins is their low solubility in physiological solutions. This renders it nearly impossible to prepare effective pharmaceutical grade injectable solutions for PDT and other applications.
Methods to prepare water soluble porphyrin derivatives for PDT are known in the art. U.S. Pat. No. 5,330,741 by Smith et al discloses a method to prepare trisodium lysyl-chlorin p6 involving the reaction between purpurin 18 methyl ester, resulting from methyl pheophorbide a transformation, and aqueous lysine in methylene chloride in the presence of pyridine. The mixture is stirred at room temperature for 12 hours, followed by the removal of the solvents in a high vacuum. The so prepared crude product is purified by reversed-phase HPLC and subsequently lyophilized. To prepare an injectable solution for the PDT of cancer, the preparation is first dissolved in phosphate buffer solution and then 0.1 N sodium hydroxide is added The pH value of the solution is adjusted to pH 7.35 using 0.1 N HCl followed by sterility filtration through a microporous filter.
Drawbacks of the above mentioned method include a lack of reproducibility and difficulty in the work-up and utilization of toxic reagents, which make it hardly appropriate for pharmaceutical manufacturing. Additionally, the prepared water soluble product of interest is stable in an aqueous solution for only 24 hours at 4xc2x0 C. in the dark, and in solid form for up to 4 months at 4xc2x0 C. in the dark [M. W. Leach, R. J. Higgins, J. E. Boggan, S.-J. Lee, S. Autry, K. M. Smith, Effectiveness of a Lysylchlorin p6/Chlorin p6 mixture in Photodynamic Therapy of the Subcutaneous 9L Glioma in the Rat. Cancer Res., 1992, 52, 1235-1239; U.S. Pat. No. 5,330,741].
There is a method to prepare a water-soluble sodium salt of pheophorbide a (3), described in U.S. Pat. No. 5,378,835 by Nakazato. According to this invention, pheophorbide a (4) is dissolved in diethyl ether, and a very diluted solution of alkali in n-propanol, iso-propanol or in their mixture is added dropwise and very slowly to the solution. The reaction is maintained up to the complete precipitation of pheophorbide a salt, which is separated by centrifugation and dried in vacuo. Then the product is dissolved in water resulting in a solution with concentration 0.5% and pH 9.2-9.5 that is then diluted with a phosphate buffer with pH 7.4-7.8.
The drawback of the method described by Nakazato is the fact that a concentrated ( greater than 1%) injectable pheophorbide a solution in water can not be generated by this technique. Additionally, the authors of the present invention demonstrated the chemical instability of such salts when stored dry, and their incomplete ability to dissolve in water after having been stored in the dry state. 
The closest analogue to the present invention is the method disclosed in Russian Patent No. RU2144538 by G. V. Ponomarev et al to prepare water-soluble complexes of chlorin e6 (7) with spacious organic amines including N-methyl-D-glucosamine by a multi-step straightforward sequence of chemical reactions including preparation of chlorophyll a from Spirulina Platensis cyanobacteria biomass, further conversion into chlorin e6 according to standard procedures [S. Lxc3x6tjxc3x6nen, P. H. Hynninen, An improved method for the preparation of (10R)- and (10S)-pheophytins a and b. Synthesis. 1983, 705-708; P. H. Hynninen, S. Lxc3x6tjxc3x6nen, Preparation of phorbin derivatives from chlorophyll mixture utilizing the principle of selective hydrolysis. Synthesis. 1980, 539-541; S. Lxc3x6tjxc3x6nen, P. H. Hynninen, A convenient method for the preparation of wet chlorin e6 and rhodin g7 trimethyl esters. Synthesis, 1980, 541-543] with an overall yield exceeding 50% after precipitation of chlorin e6 by way of stepwise addition of water to its acetone solution, followed by separation by centrifugation and 3-fold washing with water and subsequent treatment of wet chlorin e6 with water solution of 2 g-eq. spacious organic amine.
The key disadvantages of this method, which cause critical difficulties for preparative syntheses of water-soluble chlorins and particularly for industrial syntheses and drug manufacturing, are the following:
1. Chlorin e6 as an intermediate product is obtained as a wet mass with unknown definite content of chlorin e6. This instability of the amount of chlorin e6 obtained creates uncertainties that undermine the ability to standardize further resultant solutions.
2. The key intermediate in the synthetic sequence is pheophorbide a (4) which is difficult to handle for purification and standardization due to its acidic properties. Separation of pheophorbide a (4) via repeatable precipitations (as used by Ponomarev) is not quantitative and thus not convenient for large scale preparations.
3. Pheophorbide a (4) obtained by the indicated method contains impurities that are difficult to separate. This disadvantage causes uncertainty in the quantification of pheophorbide a (4) and disturbs the chemical opening of cyclopentanon ring in the course of transformation of pheophorbide a (4) into chlorins.
4. It should be noted that the samples of water soluble salts of chlorin e6 being prepared according to Ponomarev contain a variety of impurities of non-porphyrin and porphyrin types which could not be separated from the target chlorin e6 product with the use of the procedures described therein. Particularly, among the porphyrin impurities one could note by using TLC and HPLC methods are pheophorbide a (4), purpurin 18 (8), chlorin p6 (9) and some other concomitants. 
It could be noted that the compounds of types (4), (8) and (9) as salts with hydrophilic amines of the above invention are characterized by remarkably lower water solubility as compared with their respective chlorin e6 salts. Nevertheless in the presence of chlorin e6 salts the compounds of types (4), (8) and (9) as salts with hydrophilic amines of the above invention are much more water soluble than probably could be explained by possible formation of complexes with chlorin e6 salts. This phenomenon makes it impossible to separate the chlorin e6 products from impurities like the compounds of types (4), (8) and (9) by using their different water solubility.
5. The organic amines being used by Ponomarev for preparation of water-soluble chlorins are not optimal for practical applications. Particularly D-glucosamine, which forms complexes with chlorin bearing a higher solubility, is not stable enough due to possible oxidation at its aldehyde group. At the same time D-glucosamine can be present in the solution in several isomeric forms that brings structural uncertainties and therefore respective difficulties for detailed structural characterization thus failing to meet the demand of quality control for pharmaceutical preparations. One more spacious amine is used by Ponomarev, namely N-methyl-D-glucosamine, which has the same disadvantages as the above mentioned D-glucosamine and is moreover not readily available due to its difficult preparation.
6. Ponomarev claims the formation of water-soluble salts of chlorin e6 derivatives with spacious organic amines which is very uncertain because usual spacious organic amines, e.g. that ones containing tert-butyl, neopentyl, adamantyl, cyclohexyl groups, could not be used in the preparation of water-soluble chlorin e6 salts due to high hydrophobicity of spacious organic moieties.
This aspect along with other aspects mentioned above make use of the method claimed by Ponomarev impossible for manufacturing of effective pharmaceutical grade compositions according to GMP standards.
Starting porphyrin derivatives for the syntheses of interest in the present invention are traditionally obtained from pure and standard raw porphyrin materials methyl (5) or ethyl (6) pheophorbide a. General methods known to date for the separation of porphyrins from biological raw materials consist of a long sequence of laborious washings with organic solvents and/or freezing steps to destroy cell walls of the biomaterial, and repeatable extractions together with chemical treatments of the biomass to first obtain chlorophyll, which is then transformed into pheophytin and subsequently hydrolyzed to yield pheophorbide (K. M. Smith, D. A. Goff and D. J. Simpson, J. Amer. Chem. Soc., 1985, 107, 4946-4954; R. K. Pandey, D. A. Bellnier, K. M. Smith and T. J. Dougherty, Photochem. Photobiol., 1991, 53, 65-72; W. A. Svec, In: The porphyrins, ed. D. Dolphyn, NY, Academic Press, 1978, 5, 342-400).
Thus, there is a need to provide an easy and efficient method for the preparation of pure and chemically stable water-soluble pharmaceutical grade porphyrin derivatives with standard content of the desired substance, suitable for medical applications especially in photodynamic therapy. The present invention fulfills this need and further provides other related advantages.
It is an object of the present invention to provide chemically stable water-soluble porphyrin derivatives with a standard content of the desired substance and suitable for various medical applications, particularly for PDT.
It is another object of the present invention to provide pharmaceutical grade high-purity water-soluble porphyrin derivatives that are effective in pharmaceutical compositions.
It is another object of the present invention to provide a method to prepare chemically stable water-soluble porphyrin derivatives.
It is yet another object of the present invention to provide an easy and time-efficient method to prepare chemically stable water-soluble porphyrin derivatives from biological raw materials while avoiding the disadvantages of the prior art.
It is still another object of the present invention to provide chemically stable water-soluble porphyrin derivatives in a pharmaceutically acceptable preparation for use in medical applications such as treatment of cancer and other hyperproliferative diseases, infections and others.
An embodiment of the present invention consists of a method to prepare water-soluble porphyrin derivatives comprising the steps of one- or two-step direct acidic alcoholysis of biological raw material producing a crystalline alkyl pheophorbide, conversion of the obtained alkyl pheophorbide into an acidic porphyrin, and reaction of the acidic porphyrin in water or in an aqueous organic solution with a hydrophilic organic amine.
Another embodiment of the present invention consists of a method to prepare water-soluble porphyrin derivatives, comprising reaction of the acidic porphyrin in water or in aqueous organic solution with a hydrophilic organic amine.
Yet another embodiment of the present invention consists of a method to prepare water-soluble porphyrin derivatives, comprising the steps of one- or two-step direct acidic alcoholysis of biological raw material producing crystalline alkyl pheophorbide, conversion of the obtained alkyl pheophorbide into an acidic porphyrin, reaction of the acidic porphyrin in water or in aqueous organic solution with a hydrophilic organic amine, and purification of the resultant water-soluble porphyrin derivative by reversed phase chromatography using volatile solvents.
In still another embodiment, the present invention provides a method to prepare water-soluble porphyrin derivatives comprising reaction of the acidic porphyrin in water or in aqueous organic solution with a hydrophilic organic amine, and purification of a water-soluble porphyrin derivative by reversed phase chromatography with the use of volatile solvents. Furthermore, the present invention provides a water-soluble porphyrin derivative of formulae (1) and (2), useful for pharmaceutical compositions for use in photodynamic therapy and other medical applications, obtained by the methods provided by the invention.